RESEARCH & RELATED - OTHER PROJECT INFORMATION - PROJECT SUMMARY/ABSTRACT The goal of the Program Project is to explore the functional properties of the CB1 and CB2 cannabinoid receptors through the development of suitable novel ligands and by obtaining structural and functional information on these receptors. During the current cycle, we have developed high-affinity covalent and non- covalent CB1/CB2 ligands to study their foot-printing at the receptor using a Ligand Assisted Protein Structure (LAPS) approach which includes the use of covalent ligands, receptor mutants and LC/MS/MS methods. The results are further elaborated using computationally-derived receptor models. To date, our results provide initial evidence that different classes of cannabinergic ligands interact and activate/deactivate the CB1 and CB2 receptors through distinct binding motifs associated with distinct ligand-receptor conformations. In parallel with our ligand and CB1/CB2 structural work, our collaborators have provided evidence that CB1 (Bohn) and CB2 (Mackie) different agonists activate these receptors through distinct signaling pathways (functional selectivity). We have hypothesized that the individual ligand- receptor binding motifs we have identified, in turn, may be associated with distinct identifiable signaling pathways leading to different ligand-associated pharmacological profiles. A central theme for this Program Project renewal application is that distinct signaling profiles identified within CB1 and CB2 will allow us to design functionally selective ligands. This novel functional pharmacophore-based drug design should lead to new ligands with improved pharmacological profiles. This Program Project renewal proposes to: 1) design and synthesize first and later generation covalent and non-covalent ligands to be used to probe the functional properties and binding motifs of the key classes of cannabinergic ligands; 2) identify the binding motifs of the covalent ligands by using receptor mutants and LC/MS/MS methods; 3) Identify CB1 and CB2 signaling profiles of the most successful ligands; and 4) evaluate in vivo the CB1 and CB2 ligands using the mouse tetrad test as well as in animal models for neuropathic pain. The results to be obtained will serve as a basis for the development for therapies for addiction and pain.